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  • Nebulization, other shearing methods

    I cannot get dna to fragment to 200bp. I am using nebulization protocol from Illumina. I have tried 35psi 6 minutes, 35psi 8 min, 35 psi 10 min, 37psi 6 min, & 40psi 6 min. I am trying this on dna fragments ranging from 10 kb to 0.5 kb. I then run them on a 2% agarose gel and I get a smear between 300 & 500 bp. Any suggestions?
    Thanks

  • #2
    cross-post answer

    There is a google group for solexa where this same topic was addressed.



    Here's the thread and response:



    I was in the same situation. I never did get nebulization to work the
    way Illumina says it will. After 2 months of troubleshooting their
    protocol, doing many of the same things you've done as well as some
    other things, it never worked. I submitted to Sonication. I used both
    the Sonicator 300 and the Bioruptor. I found that with the Bioruptor
    you can do many 1.5mL tubes at one time without contamination being an
    issue. (consistancy is also better) The Sonicator 300 was a single
    probe that could potentially cause contamination. If you would like
    information on the Bioruptor I can get it for you. I'm sure other
    sonicators would work just as well just be careful of single probe
    sonicators unless you have a fool proof cleaning method. I brought up
    5ug of DNA in 50ul of TE then added 150 ul of Illumina's Nebulization
    buffer to the tube. I sonicated it on High for 30 sec ON and 30 sec
    OFF for a total of 30 min and then cleaned it with a Qiaquick protocol
    that follows nebulization in the Genomic Prep protocol from Illumina.
    You should find that sonication will give the correct sizes. If you
    would like more info feel free to email me. Hope this helps!

    Comment


    • #3
      We've also been looking at this particular problem. At the moment we're trying to decide between the bioruptor (5-10k euros) and the covaris S2 (40-45k euros). The bioruptor seems to be capable of shearing down to 200bp while the covaris can get down to 70bp. Are there specific protocols that require DNA fragmented to 60-70bp? Or are the 200bp fragments sufficient for Solexa purposes?

      Comment


      • #4
        Hi this is Kendra~ My posting from the solexa group on google was posted here by mgogol. I just wanted to add one thing about the Bioruptor. To my knowledge there is no reason to shear DNA to 70bp. I have smears from 800-100bp from using the Bioruptor. If you have any other questions please let me know.
        Thanks!

        Comment


        • #5
          I do think there is reason to shear DNA as short as 70bp. Near target problem of nimblegen microarray-based enrichment adds to sequencing overhead. Shorter DNA would alleviate the near target problem and make enrichment more specific. We also know many exons (~40% maybe) are shorter than 100bps.

          Comment


          • #6
            I can think of at least on reason to sheer that small: in theory, the shorter your fragments are in an Illumina Chip-Seq experiment, the cleaner your peaks will be.

            OTOH, the protocols don't specify that you need 70bp fragments, and I'm not sure how well this would work in practice. (Usually 100-300bp sizes are used). I'd love to see some results with fragments that small.
            The more you know, the more you know you don't know. —Aristotle

            Comment


            • #7
              Actually what Illumina recommends for ChIP-seq is 200 bp, so it may be that this is the optimal length for the machine. Bioruptor is certainly capable of generating fragments below 200 bp, but perhaps not below 100? Anyway the 35 k€ difference would give you something like 50 extra lanes in reagents which may make up for the difference...

              Comment


              • #8
                While it's true that they recommend 200bp, I suspect that that refers to the ideal cluster density forming size, as opposed to the ideal size for locating DNA binding sites in a ChIP-Seq experiment.
                The more you know, the more you know you don't know. —Aristotle

                Comment


                • #9
                  Any further updates on shearing methods? I'd be interested to hear how many people use nebulisation versus sonication for production of small DNA fragments. Does anyone here use the nebulisers supplied with the Illumina kits?

                  We're just about to get our machine running, and we want to know whether it's worth buying a Covaris instrument.

                  Cheers,

                  Scott.

                  Comment


                  • #10
                    We just got a Covaris and a Hydroshear, I should be able to report on their respective output soon.

                    We tested a hydroshear set to shear to "2-3kb" and a surprisingly large percentage of the DNA was in that range. Didn't get a gel pic from that test though...

                    Comment


                    • #11
                      Hi ECO,

                      That'd be good. I know the Covaris machines do a great job, and I've seen a few results from these machines before.

                      What are your main applications? Which technologies? Are you using this DNA on an Illumina instrument?

                      Cheers,

                      Scott.

                      Comment


                      • #12
                        Im a little out of date on this, but generally you can grow clusters with a variety of fragment sizes. It used to be the case that shorter was better, giving small uniform and bright (efficiently amplified) clusters - and thus you can pack them in at higher densities giving higher yields (we've reached 5G PF). Longer inserts gave larger weaker clusters. The spread of your insert size also used to matter, in that if you had a broad range of sizes you'd get a broad range of cluster sizes (brightnesses) and thus your output data would show a similar range of quality. So having a tight size distribution is desirable. If you go too short, you can end up sequencing right through the inserts and into the primer/adapters (this will affect your downstream analysis and these will look like errors).

                        Comment


                        • #13
                          Originally posted by ScottC View Post
                          Any further updates on shearing methods? I'd be interested to hear how many people use nebulisation versus sonication for production of small DNA fragments. Does anyone here use the nebulisers supplied with the Illumina kits?

                          We're just about to get our machine running, and we want to know whether it's worth buying a Covaris instrument.
                          If you've ever nebulized more than one sample, you'll hate life. Long times, lots of loss, "fogging" your lab/fumehood with your sample, I'll never do it again .

                          I spent today playing with different conditions on the Covaris S2 (beads, % glycerol, etc), and the best I could do was ~50-60bp at the low end, and ~400 on the high end. In my hands, there doesn't seem like there is much variation to be had with any parameter except time. It couldn't be easier to do, with no worries about aerosols or probe contamination.

                          ABI's original protocol was 40 minutes with different conc's of glycerol...now it's 10 min with no glycerol and 2um beads. I plan to go a lot longer than 10 minutes next week to see how small I can go with the beads. The SOLiD wants 60-110bp for frag libraries and the majority of my fragments were larger than that (~250-350 was probably >50%)

                          Would love to hear other's experiences.

                          Comment


                          • #14
                            Originally posted by ECO View Post

                            ABI's original protocol was 40 minutes with different conc's of glycerol...now it's 10 min with no glycerol and 2um beads. I plan to go a lot longer than 10 minutes next week to see how small I can go with the beads. The SOLiD wants 60-110bp for frag libraries and the majority of my fragments were larger than that (~250-350 was probably >50%)

                            Would love to hear other's experiences.
                            I just listened to a seminar from a guy from the Broad who commented that they used to use beads, but found that it was the volume, not the beads themselves, that made all the difference, and now they don't use them. Don't know if that's of any help/interest, but you just reminded me when you mentioned them.

                            Scott.

                            Comment


                            • #15
                              That may be helpful...happen to remember the correlation of volume-shearing?

                              Thanks scott.

                              Comment

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